During the tenure of this K23 Award, Dr. Samuel will pursue the mechanism of fat-induced hepatic insulin resistance. He will develop the skills for in vivo human metabolic studies through didactic classes and practical experience. Under the guidance of expert mentors, he will receive training in NMR spectroscopy and GC/MS metholdogy. With these techniques, he will explore the relationship between hepatic fat accumulation, insulin resistance and type 2 diabetes mellitus, conditions that are increasingly common in American. Type 2 diabetes mellitus affects approximately 6% of the population and costs $100 billion dollars annually. Although hepatic insulin resistance is a cardinal feature of this disease, its mechanism is undetermined. There is growing appreciation that many patients with type 2 diabetes mellitus hae hepatic fat accumulation. Given that muscle fat accumulation causes peripheral insulin reisstance, we hypothesize that hepatic fat accumulation leads to hepatic insulin resistance. We predict that hepatic fat accumulation will impair the insulin signaling cascade by limiting the ability of IRS2 to activate PI3 kinase. This will impair the ability of insulin to stimulate glycogen synthesis. We also hypothesize that hepatic fat accumulation will lead to increased gluconeogenesis as a result of increased pyruvate carboxylase activity and increase transcription of PEP-CK. Both are key regulatory enzymes in gluconeogenesis. We will tests these hypotheses, lean subjects consuming a high fat diet. We predict that fat will accumulate within the liver prior to the muscle. With [13C] NMR measures of glycogenolysis, gluconeogenesis and hepatic insulin sensitivity. Through a complementary rat model of fiet induced hepatic steatosis we can examine changes in the signaling cascade, enzyme activity and gene transcription. Using adenoviral transfer of the UCP2 gene into rat livers, we will determine if increased uncoupling prevents diet induced hepatic steatosis and insulin resistance. Finally, with a novel method to directly measure gluconeogenesis, we will determine if hepatic fat accumulation increases hepatic gluconeogenesis.